Method for preventing the adhesion of particles

ABSTRACT

A method for the prevention of the adhesion of particles, in particular cells and cellular components in solution to surfaces, characterized in that to the solution is added at least one polyalcohol.

[0001] The invention relates to a method for the prevention of theadhesion of particles, in particular of cells and cellular components,to surfaces, whereby a polyalcohol is added to the solution whichcontains the particles.

[0002] The adherence to surfaces is a fundamental property of cells.This becomes apparent, for example, in the interaction of cells with anextracellular matrix, which is an important factor in controlling thegene expression or general cell function. These processes are influencedin particular via proteins on the cell surface.

[0003] Indeed, this property of cells often presents a big problemduring the preparation of cell cultures and the nondestructiveexamination of cells or cellular components. An adherence of cells to,for example, surfaces of the accessories and containers used in thepreparation is undesirable. Technologies for the microanalysis ofbiological samples in particular can not be utilized for cellexaminations without problems. These systems that contain, for example,canal systems like in the “Lab-on-Chip”-technology, often only havedimensions of a few micrometers. Furthermore, the selection of thesurface materials to be used of these microanalysis systems is bound tothe particular manufacturing process or the material of themicrostructures in itself. Microchannel analysis systems, for example,are made from glass, silicon, silicone, and organic polymers, such asPMMA or polyurethane, all substrates that facilitate the adhesion ofcells. The surface material consequently can not be freely chosenaccording to the requirements for a minimal cell adhesion.

[0004] In order to prevent cell adherence/adhesion, the surface of themicrostructures in itself is therefore modified. Various chemicalcompounds, in general hydrophobic silanes and hydrophilic polymers(hydrogels) are used for this purpose.

[0005] Carlson et al. ((1997): “Self-Sorting of White Blood Cells in aLattice” in “Physical Review Letters” 79, No. 11, p. 2149-2152)succeeded with the known coatings in the sorting of blood cells, forexample, in mechanical microfilter systems. But white blood cellsthereby irreversibly adhered to the entrances of microchannels. This isdesirable in the described system, but at the same time shows thepresent limitations in the use of microchannels for the nondestructiveexamination of cellular samples.

[0006] Further investigations by Li et al. (“Transport, Manipulation,and Reaction of Biological Cells On-Chip Using Electrokinetic Effects”in “Analytical Chemistry” (1997) 69, 1564-1568) showed that blood cellscan be brought into 40 μm-wide microchannels. But the cells weresubsequently lysed with a reagent and only then subjected to ananalysis. However, the recovery of eukaryotic, particularly of livingmammalian cells, from microchannels makes high demands on the selectionof the surface materials of the channels.

[0007] Further investigations, for example the coating of glass laminawith polyvinyl alcohol, showed that such a surface modification canminimize an adhesion of mouse fibroblast (Hisada, T. (1976): “Theadhesion of culture cells to some polymers” (Japanese language) in“Shika Rikogaku Zasshi” 17(38), p. 91-101)).

[0008] It could likewise be shown, that copolymer particles frompoly(methyl methacrylate) and polyvinyl alcohol do not adhere to bloodcells and are not phagocyted by monocytes and neutrophiles (Ayhan, H.and E. Piskin (1997): “Interaction of activated leukocytes withpolymeric microspheres” in “International Journal of Artificial Organs”20(12), 704-707).

[0009] Krylov et al. (in “Electrophoresis”, 21, 767-773, 2000)furthermore found, that the adherence of cells to glass or polystyreneis markedly reduced, if the surfaces are modified with hydrophilicpolymers (hydrogels) such as polyvinyl alcohol.

[0010] However, in all the mentioned investigations, it is necessary tocarry out a direct surface coating, whereby, for example, polyvinylalcohol (PVA) is immobile bound to the surface, that is a surfacecoating or a copolymerization of a substrate with PVA is carried out.For the implementation of the coating, complicated, in particular toexact temperature specifications bound, multistep and thereby verytime-consuming processes are necessary. Furthermore, particularly thecoating of surfaces that consist of polymers such as polystyrene isproblematic, since this material can be damaged by the temperatures offar above 100° C. that are necessary for the coating. The method cantherefore only be satisfyingly applied to the use with glass surfaces.

[0011] Furthermore, it is often impossible, in particular due to theonly small diameters of the microstructures in microanalysis systems, toachieve a coating that is satisfying. Thus, for example, a uniformdistribution of the polymers or other coating materials across thesurfaces of the corresponding microstructures, for example of channelstructures of a diameter of only a few micrometers, is not or onlyinsufficiently possible.

[0012] It is therefore the object of the present invention to develop amethod, that independent of the nature and surface properties of amicroanalysis system allows an essentially nondestructive and loss-freeexamination of particles, particularly of cells and cellular components,in solution in these systems.

[0013] The object is solved by a method according to claim 1 and asolution according to claim 12.

[0014] The method comprises according to the invention the addition of asubstance to the solution that contains the particles, particularlycells or cellular components, but also, for example, may containproteins and peptides, that are present in the free form or bound tostructures such as, for example beads. This substance according to theinvention causes the adherence of these particles to surfaces,particularly surfaces of a microanalysis system, to be essentiallyprevented.

[0015] In the only figure, a diagram is shown that illustrates therecovery rate of suspended Jurkat cells from a microliter-syringe versusthe residence time t in the presence of 0.5% PVA.

[0016] According to the invention this substance involves a polyalcohol,in particular polyvinyl alcohol, for example polyvinyl alcohol VA30,000-70,000 and/or polyglycol, preferably polyethylene glycol.Surprisingly, the addition of this substance to the solution did notshow a toxic effect relating to living cells or does not cause change ofcellular components. Especially suitable is a concentration of thepolyalcohol in solution of 0.01% (w/v) to 5% (w/v). Consideringpolyvinyl alcohol as an example, it could even be shown, that theanti-adhesion effect of the polyalcohol by far exceeds that of knownsubstances that are used as anti-adhesives (table 1). The watersolubility and additional properties, listed below, of the polyalcoholcause a universal applicability in all aqueous particle solutions.

[0017] An additional important property of this substance, that inparticular qualifies it for the use in solutions which contain cellsand/or cellular components, is that it has no influence on thephysiologic pH-value of the solution.

[0018] Many methods of analysis, that are applied in microanalysissystems, are based upon the recording and evaluation of optical signals,such as, for example, fluorescence correlation spectroscopy,fluorescence resonance energy transfer, fluorescence polarization,fluorescence intensity distribution analysis, or, for example,UV-spectroscopy. Therefore, it was all the more important for theuniversal applicability of this substance, that it does not influence anoptical analysis, i.e. particularly does not show autofluorescence orchange the refractive index of the solution. These requirements are verywell met by polyalcohols, particularly polyvinyl alcohols orpolyglycols.

[0019] The method according to the invention now allows an essentiallyloss-free examination and manipulation, particularly separation, of theparticles, particularly in microanalysis systems.

[0020] Hereby it may concern microanalysis systems that have, forexample, channels with a height of from 1-1,000 μm, particularly 2-500μm, especially preferred 40 μm; a width of 100-2,000 μm, particularly200-600 μm, and a length of 100 μm-10 cm, preferably 1-2 cm. The systemsmay consist in particular of glass, silicon, silicone, organic polymers,or another suitable material. Specific embodiments of the microanalysissystems may also have electrodes, in particular for the generation ofdielectric forces for the manipulation of the particles that are to beexamined, for example for the microscopic, optical and/or electricalanalyzing, for the sorting, separating, electroporating, fusing andseparating of the particles, particularly of cells.

[0021] Microanalysis systems, for example, thus comprise electrodes asdielectric junctions in the microchannels for the sorting of suspendedparticles (in particular cells), or in other microanalysis systems theelectrodes are arranged in such a way that the cell fusion and/or cellporation can be carried out, and other systems comprise two electrodelevels in a closed microchannel, a dielectric field cage in a channelintersection as well as electrodes developed as dielectric junctions forthe implementation of a stop-flow analysis as well as a subsequentsorting of the analyzed particles. Whereby, the microchannel systems mayalso comprise combinations of the various electrode systems.

[0022] Even the injection of the particles into such a system, which isoften carried out with the help of a plastic or glass syringe, does nolonger lead to losses, which are caused by the adherence of theparticles to the syringe body.

[0023] Since the added substance according to the invention also doesnot influence a living cell, it is, for example, possible, to directlyexamine cells, to sort on the basis of the examinations and then tospecifically recreate cell cultures with the separated cells. That way,for example, cells with a certain property, for instance with a certainkind of receptors on the cell surface, are specifically and essentiallyloss-free separated from other cells and multiplied.

[0024] The method according to the invention is in general suitable forall cell types, consequently both for eukaryotic cells and forprokaryotic cells.

[0025] In table 1, the portion of living cells after the examinationand/or manipulation in various microanalysis systems in the presence ofpolyvinyl alcohol is shown. (Jurkat-cells in Cytocon™ sorter chips (No.1 and 2), in Cytocon™ porator chips (No. 3-5), and Cytocon™ loader chips(No. 6 and 7)). TABLE 1 Flow Rate in μl/h Sheath Cell Amount RecoveryNo. Sample fluid Per μl Total Rate % 1 10 288 660 440 84 2 10 288 132220 64 3 10 288 689 74 4 10 144 144 100 5 10 144 144 113 6 18 144 160160 87.3 7 20 18 305 243 63

[0026] In table 2, in each case, the portion of Jurkat-cells and bloodcells in the fractions 1 and 2 after a cell separation in amicroanalysis system (Cytocon™ chip) is shown. TABLE 2 Number of CellsTotal Fraction 1 Fraction 2 Total Jurkat RBC Total Jurkat RBC TotalJurkat RBC 6,355 161 6,194 345 151 194 6,010 10 6000 100% 2.5% 97.5%100% 43.8% 66.2% 100% 0.17% 99.8%

[0027] The method according to the invention is likewise suitable forcellular components. It has no influence on their structure and does notcause degeneration. Under cellular components are to be understood, forexample, liposomes, lipid membranes, lipids, proteins, DNA, nucleicacids, messenger substances, sugar, glycans, and/or glycoproteins. Inprinciple, all components of a cell are thus concerned, but also cellfragments.

[0028] The method according to the invention provides, besides theadvantage that it prevents an adherence of the particles to the surfacesof microanalysis systems, that may in particular consist of silicon,silicone, glass, and/or organic polymers such as PMMA, polyurethane,polycarbonate, polypropylene, polystyrene, polyethylene, polyvinylchloride, Teflon®, polyacryl (fiber), nylon®, and/or perlon®, also thepossibility that it prevents the adhesion of biomolecules, in particularcells, cell membranes, cellular components such as lipids, proteins (inparticular antibodies), DNA, nucleic acids, messenger substances,biotin, sugar, glycans, and/or glycoproteins among one another or tosurfaces coated with such molecules.

[0029] In table 3, the cell adherence to polystyrene culture dishes inthe presence of various substances is shown. TABLE 3 Jurkat U937Adherence Vitality Adherence Vitality RPMI —  97%  9 100%   10% FBS PBS999  99% 999  98% PBS + Ca, Mg 999 OK 999 OK  0.1% BSA  99 OK 999 OK 0.5% BSA — OK  99 OK   1% PVP 15  99 100% 999 100%   1% PVP 25  99 100%999 100%   1% PEG 5,000  99 100% 999 100%   1% MC 15  99 OK 999 OK   1%MC 400  99 OK  99 OK   10% Ficoll 70 999 OK 999 OK   10% Ficoll 400 999OK 999 OK   10% 999 OK 999 OK Dextran T10   1% PVA —  97% (9)  98%30,000-70,000  0.1% PVA — OK (9) OK 30,000-70,000 0.01% PVA — OK (9) OK30,000-70,000

[0030] Whereby within the meaning of this invention by surface is meantany surface, but in particular channel surfaces, surfaces of storagecontainers of microanalysis systems, tubing, syringes, injectionmodules, or surfaces of synthetic microparticles.

[0031] Furthermore, a solution was found, that in general is suitablefor the examination of particles, in particular of cells and cellularcomponents in microanalysis systems. This solution contains salinebuffer, in particular PBS, and/or inositol, and polyvinyl alcohol.However, to the solution may also be added other components known in thecontext of cell solutions such as, for example, magnesium and calciumions.

[0032] The portions of saline buffer, inositol, and/or PVA are variable.The buffer may be diluted in a variable mixing ratio by normoosmolar,hyperosmolar, or hypoosmolar sugar solutions (for instance sucrose orinositol). The portion of saline buffer in the solution may be variedbetween 0 and almost 100%, whereby portions of 20%, 50%, or almost 100%are especially preferred.

[0033] Inositol is preferably used as a 0.2 to 0.5 M solution, forinstance of inositol in distilled water, especially preferred a 0.3 Msolution. Whereby, the portion of inositol solution and the solutionaccording to the invention may also be varied between 0 and almost 100%.Especially preferred are indeed portions of 50%, 80%, and almost 100%.

[0034] Polyvinyl alcohol with a molecular weight of between 30,000 g/moland 70,000 g/mol is particularly well suited for the solution accordingto the invention, whereby the amount of PVA in the solution according tothe invention lies between 0.1% (w/v) and 1.1% (w/v), preferably between0.3% (w/v) and 0.6% (w/v), especially preferred between 0.45% (w/v) and0.55% (w/v).

[0035] The solution according to the invention may be utilizedindependent of type and properties of the particles, in particular fortheir examination and manipulation in microanalysis systems.

[0036] For this purpose, the particles may be directly suspended in thissolution and placed in the microanalysis system. However, it is alsopossible to add this solution to particle suspensions.

EXAMPLE 1

[0037] Influence of Polyvinyl Alcohol on the pH-Value of a Solution

[0038] The pH value of a 1% solution of polyvinyl alcohol in PBS wasmeasured and lies at 7.31 (the physiologic pH range is pH 7.2-7.4).

EXAMPLE 2

[0039] The Influence of Polyvinyl Alcohol on the Cell Adhesion inComparison to Other Additives

[0040] Solutions:

[0041] Jurkat-cells (clone E6-1 from the European Collection of AnimalCell Cultures, Salisbury, England) and U937-cells (monocytic cell lineof the European Collection of Animal Cell Cultures, Salisbury, England)were cultivated in RPMI 1640 medium (GIBCO Life Technologies, Karlsruhe)by adding 100 IU/ml each of penicillin and streptomycin(Seromed/Biochrom, Berlin) and 10% fetal calf serum (Seromed/Biochrom,Berlin). 3 ml each of the cell suspension were centrifuged off and takenup in 300 μl of phosphate-buffered saline solution without calcium ormagnesium (PBS, Seromed/Biochrom, Berlin). The wells of a 24-well plate(24-well polystyrene culture plates, Corning Costar) were each filledwith 500 μl PBS by adding the in table 1 indicated concentrations of thesubsequently listed substances. Methylcellulose (15 and 4,000 centipoiseviscosity of a 2% solution, SIGMA Aldrich GmbH, Steinheim), Ficoll 70and Ficoll 400 (Pharmacia, Uppsala, Sweden), Dextran T10 (Pharmacia,Uppsala, Sweden), polyvinyl pyrrolidone 15 and 25 (Serva), polyethyleneglycol 5,000 (Fluka) and polyvinyl alcohol 30,000-70,000 SIGMA AldrichGmbH, Steinheim) were tested. 50 μl of the cell suspension describedabove were added. A sample in 500 μl cell culture medium (in whichadherence of these suspension cells does not occur) served as control.

[0042] Implementation:

[0043] The samples were incubated for 30 minutes at 37° C. Thesupernatant with non-adhered cells was removed and replaced by buffer.The adherence was qualitatively evaluated (refer to table 1). The cellsin both supernatant and the wells were stained with trypanblue solution(0.2%, SIGMA Aldrich GmbH, Steinheim) in order to examine the vitalityof the cells. Only polyvinyl alcohol has a very distinct inhibitoryeffect on the cell adherence with both cell lines. The effect alreadyoccurs at a concentration of 0.1% (w/v).

[0044] Result:

[0045] Many substances, to which an anti-adhesive effect is assigned,were tested in comparison to polyvinyl alcohol. Bovine serum albumin(BSA), for instance, which is known from flow cytometry for the blockingof non-specific bonds and used in immunoreactive detection (e.g. Westernblot technique). Furthermore, various hydrophilic polymers that are usedin density gradient centrifugation of blood samples or in cell culturetechniques were examined. Even the influence of cadherins and integrins,specific cell-cell and cell-substrate adhesion molecules, in bufferswithout calcium and magnesium were examined. These molecules needcalcium for interaction. It became apparent, that polyvinyl alcoholdistinctly surpasses the other substances in the effect of successfulpreventing an adhesion of suspended cells on the sample carrier.

EXAMPLE 3

[0046] Examination of the Toxicity of Polyvinyl Alcohol with Respect toLiving Mammalian Cells

[0047] Implementation:

[0048] The use of 0.5% (w/v) of PVA for the recovery of cells from amicroliter syringe was examined. Jurkat-cells were suspended in aconcentration of 5.69×10⁵ cells per ml in PBS with 0.5% PVA (w/v). 5 μlof this suspension were drawn up into a 5 μL-syringe (Dynatech). 1 μleach were transferred immediately and after 1 and 5 minutes,respectively, into a well of a Terasaki plate (NUNC, Wiesbaden). Thecells were stained in the plate with the live/dead stain kit (MolecularProbes, Leiden, Netherlands) and counted; thereby their vitality wassimultaneously examined. The recovery rate was determined.

[0049] Result:

[0050] More than 90% of the cells could be recovered—after 5 minutes aswell—from the syringe. They therefore had not adhered. The percentage ofdead cells initially, thus at 0 minutes, amounted to 0.8% and increasedafter 5 minutes only to 2.4%.

EXAMPLE 4

[0051] Examination of Cells in Microanalysis Systems Using a Cell Bufferwith Polyvinyl Alcohol

[0052] Material:

[0053] The use of polyvinyl alcohol for the work with suspended cells inmicrochannels was examined with a Cytocon™ 300 device system. TheCytocon™ chips consist of glass and exhibit channels with a height of 40μm, a width of 200-600 μm, and a length of about 1-2 cm, wherebyspecific embodiments of the chips may also exhibit electrodes in thechannels. Cytocon™ sorter chips comprise, for example, electrodes asdielectric junctions in the microchannels for the sorting of thesuspended particles (in particular cells), in Cytocon™ porator chips theelectrodes are arranged in such a way, that cell fusion and/or cellporation can be carried out, and Cytocon™ loader chips, that exhibit twoelectrode levels in a closed microchannel, a dielectric field cage in achannel intersection, as well as electrodes developed as dielectricjunctions, are suitable for the performance of a stop-flow analysis aswell as the subsequent sorting of the analyzed particles.

[0054] Polyvinyl alcohol with a molecular weight of 30,000 g/mol to70,000 g/mol was used. The polyvinyl alcohol was dissolved in the bufferto 0.5% (w/v) and was present during the entire manipulation of thecells in the microchannels.

[0055] Implementation:

[0056] The cells were suspended in the buffer and injected into aCytocon™ chip. The suspension was transported within the channel systemwith a certain flow rate (“sample”) and at the chip exit acceleratedwith a sheath fluid. The rinsed out cells were counted and the recoveryrate calculated from the initial concentration and the injected sampleamount.

[0057] Result:

[0058] Even though the cell amount used was only a few hundred cells andthe flow rate in the microchannels was very slow (dwell time of thecells in the microchannel about 2 minutes), the recovery rate of cellswas very high (table 2) and values around 100% were achievable.

EXAMPLE 5

[0059] Fractionation of Blood Cells (Separation of Lyphocytes from RedBlood Cells)

[0060] Implementation:

[0061] A mixture of whole human blood, Jurkat T-lymphoma cells, and aPVA-containing buffer was prepared. The buffer contained 20% (v/v) PBS,80% (v/v) 0.3 M inositol solution, and 0.5% (w/v) PVA (30,000-70,000,Sigma). For a better visualization during the experiment, the Jurkatcells were labeled with 10 μM Calcein-AM™ (Molecular Probes).

[0062] The blood and the lymphoma cells were diluted as follows: Thecell samples were mixed to concentrations of 2.5·10⁶ Jurkat cells/ml and1.8·10⁷ red blood cells/ml. Samples of 0.3 μl were injected into aCytocon™ chip (microanalysis system).

[0063] The Cytocon™ chips used for this experiment consisted of glassand exhibited channels with a height of 40 μm, a width of 200-600 μm,and a length of about 1-2 cm. Additionally, the microanalysis systemused here, comprised electrodes in one or several microchannels, thatare arranged here as dielectric junction for the sorting of the cells.

[0064] The cells were separated based on differences in their size andtheir dielectric properties via the so-called switch electrode(junction) of the Cytocon™ microanalysis system at a frequency of theelectric field of 800 kHz and an amplitude of 3-6 V rms at a flow rateof 62-240 μm/s. Under these conditions, the Jurkat cells were deflectedand collected in fraction 1, while the red blood cells (RBC) could passthe junction and were collected in fraction 2.

[0065] Jurkat cells and red blood cells (RBC) were counted in bothfractions in order to be able to determine the accumulation and theyield.

[0066] Result:

[0067] The accumulation factor for the Jurkat cells in fraction 1 is inan exemplary experiment 17.5. The yield of Jurkat cells in this fractionwas 93% in this case (table 3).

[0068] Overall (fractions 1 and 2), the cell recovery rate was almost100%.

1. A method for the prevention of the adhesion of particles, inparticular cells and cellular components in solution to surfaces,characterized in that to the solution is added at least one polyalcohol.2. The method according to claim 1, characterized in that theconcentration of the polyalcohol in solution is 0.01% (w/v) to 5% (w/v).3. The method according to claim 1 and/or claim 2, characterized in thatthe polyalcohol is water-soluble.
 4. The method according to at leastone of the claims 1 to 3, characterized in that the polyalcohol ispolyvinyl alcohol or polyglycol, in particular polyethylene glycol. 5.The method according to at least one of the claims 1 to 4, characterizedin that the cells are eukaryotic and/or prokaryotic cells.
 6. The methodaccording to at least one of the claims 1 to 5, characterized in thatthe cellular components involve liposomes, lipid membranes, lipids,proteins, DNA, nucleic acids, messenger substances, sugar, glycans,and/or glycoproteins.
 7. The method according to at least one of theclaims 1 to 6, characterized in that the surfaces involve cells, cellmembranes, cellular components, uncoated or with biomolecules coatedsurfaces from silicon, silicone, organic polymers, and/or glass.
 8. Themethod according to claim 7, characterized in that the biomolecules arelipid membranes, lipids, proteins (in particular antibodies), DNA,nucleic acids, messenger substances, biotin, sugar, glycans, and/orglycoproteins.
 9. The method according to claim 7, characterized in thatthe organic polymers are PMMA, polyurethane, polycarbonate,polypropylene, polystyrene, polyethylene, polyvinyl chloride, Teflon®,polyacryl, nylon®, and/or perlon®.
 10. The method according to at leastone of the claims 1 to 9, characterized in that the surfaces involvechannel surfaces of a microanalysis system.
 11. The method according toat least one of the claims 1 to 10, characterized in that the surfacesinvolve surfaces of synthetic microparticles.
 12. A solution containing:saline buffer, inositol, polyvinyl alcohol, in particular for the use inmicroanalysis systems.
 13. The solution according to claim 12,characterized in that the saline buffer is PBS.
 14. The solutionaccording to claim 12 and/or claim 13, characterized in that the portionof saline buffer and/or inositol is variable.
 15. The solution accordingto at least one of the claims 12 through 14, characterized in that theamount of PVA lies between 0.1% (w/v) and 1.1% (w/v), preferably between0.3% (w/v) and 0.6% (w/v), especially preferred between 0.45% (w/v) and0.55% (w/v).